Serotonin (5-HT) and Brain derived neurotrophic factor (BDNF) are two signaling molecules that play important regulatory roles in the development and plasticity of neural circuits that are known to be altered in psychiatric disorders including depression. The role of BDNF signaling in depressive behavior is substantiated by studies showing decreased levels of BDNF and its receptor, TrkB in postmortem brain as well as peripheral tissues of depressed subjects. In addition, increases in extracellular 5-HT have been shown to enhance BDNF signaling in rodents. However, the mechanism by which 5-HT regulates TrkB signaling is poorly understood. The overall goal is to determine the role of transglutaminase 2 (TG2) in regulation of TrkB signaling by 5-HT. TG2 is a calcium-dependent enzyme that plays an important role in posttranslational modification of proteins. The role of TG2 in transamidation of 5-HT to Rac1 has recently been reported in cortical neurons. Rac1 is a member of the Rho family of small GTPases involved in signal transduction pathways and is known to promote TrkB endocytosis. We hypothesize that increased TG2-dependent transamidation of 5-HT to Rac1 inhibits TrkB signaling with depressive phenotype in mice. We provide initial evidence demonstrating that mice with neuronal TG2 overexpression (TG2+/+) show depressive phenotype with reduced Rac1 protein levels in the frontal cortex. We found reductions in TrkB signaling, Hap1 (a huntingtin associated protein involved in intracellular trafficking of endocytic TrkB) levels and its association with TrkB in frontal cortex of TG2+/+ mice as well as in long-term 5-HT-treated primary cortical neurons. We will also investigate the role of Hap1 in mediating Rac1-dependent TrkB endocytosis. We found that inhibition of TG2 activity attenuates 5-HT-induced Rac1 degradation and association of 5-HT with Rac1, but increases the association of Hap1 and TrkB in cortical neurons. Moreover, increases in TG2 protein, but decreases in TrkB, Rac1 and Hap1 levels, were found in prefrontal cortex of suicide subjects. These data suggest that Rac1 and Hap1 function downstream of TG2 and are involved in linking 5-HT to TrkB signaling. The specific aims are designed by using pharmacological and genetic approaches for TG2 in in vitro and in vivo models to provide a comprehensive series of studies aimed at examining the role of TG2 and Hap1 in TrkB signaling by 5-HT. Three Specific Aims are proposed. The Specific Aim 1 will test the hypothesis that that TG2-dependent transamidation of 5-HT induces depressive phenotype in mice. The Specific Aim 2 will test the hypothesis that that impaired TrkB signaling contributes to the depressive phenotype seen in TG2 overexpressed mice. The Specific Aim 3 will test the hypothesis that Hap1 mediates Rac1-dependent impaired TrkB endocytosis. Given the important role of TrkB in neuroplasticity, identifying novel regulatory mechanisms of TrkB by 5-HT may provide avenues to develop newer therapeutics for depression and related psychiatric disorders.